Epstein-Barr virus (EBV) is an extremely successful pathogen, being able to establish a lifelong latent infection within B lymphocytes of its human host, with overt disease in healthy individuals restricted to a self- limiting mononucleosis in ~40% of individuals when infection is delayed until the second decade of life. However, a breakdown in cellular immunity, particularly as a consequence of AIDS, remains a significant risk factor for development of EBV-associated lymphoma and lymphoproliferative disease, underscoring the highly evolved equilibrium that exists between EBV and its host. Establishment of this equilibrium, and EBV's oncogenic potential are dependent on a highly coordinated and controlled expression of the EBV latency- associated genes, which encode not only proteins but at least four classes of noncoding RNAs (ncRNAs). The long-term objective of the research proposed in this application is to understand the contributions made to EBV infection and pathogenic potential by two EBV RNAs belonging to the class of long ncRNAs (lncRNAs), i.e., RNAs longer than 200 nucleotides with little or no functional protein-encoding capability. These lncRNAs are encoded by the paralogous EBV genes BHLF1 and LF3, long known to be expressed at high level during the virus replication or lytic cycle, but recently identified as beig actively expressed during latent infection as well. Our preliminary results indicate that, upon deletion of the BHLF1 locus from the EBV genome, the virus is unable to sustain full expression of the EBV latency-associated proteins, converting to a more restricted program of latency-gene expression associated with long-term EBV persistence within its cellular reservoir, the memory B cell. Thus, we hypothesize that the BHLF1 lncRNA, and its paralog from LF3, play active but distinct roles in the regulation of EBV latent infection and persistence. We propose four specific aims: Under Aim 1, we will identify proteins that interact with the BHLF1 lncRNA, and the role that these specific RNA:protein associations play in EBV latent infection. In Aim 2 we will elucidate the expression patterns of the BHLF1 lncRNAs during the different EBV latency programs (defined by specific patterns of viral gene expression), and whether they traffic to specific subcellular domains with known functionality, possibly revealing function(s) of the EBV lncRNA. In Aim 3 we will elucidate whether the BHLF1 gene locus itself, in concert with its lncRNA, acts as a central determinant of EBV latency program by regulating viral genome configuration and other mechanisms. Finally, under Aim 4 we will begin to define the roles that the LF3 locus and lncRNA contribute to EBV latency, taking a similar tack as we have proposed for BHLF1.